兰州大学机构库 >学院待认领
MSCs的标记跟踪及其移植后对大鼠失神经骨折病理性骨痂塑形影响的实验研究
Alternative TitleThe influence of MSCs autologous transplantation in denervated fracture healing:An experimental study in rats
雷栓虎
Thesis Advisor汪玉良
2013-05-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword骨髓间充质干细胞 失神经 骨折愈合 表面标志物 细胞染色
Abstract目的:研究大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)体外分离培养和染色标记,同时利用细胞标记技术探讨BMSCs移植对失神经骨折愈合骨痂改变的影响。 方法:骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)取自SD雄性大鼠(约80 g)的股骨和胫骨,采用贴壁分离培养法培养BMSCs,同时流式细胞仪对其表面抗原(CD34, CD44,CD45) 进行测定;然后分别用BrdU、DAPI 以及Hoechst33342对BMSCs进行染色标记,观察三种不同方法体外染色的合适时间和最佳剂量,同时MTT检测细胞增值率。上述实验完成后,选取3月龄雄性SD大鼠48只随机分为6组(n=8),A组为左胫骨骨折组,B组为T10脊髓横断+左胫骨骨折组,C组为T10脊髓横断+左胫骨骨折+自体骨髓间充质干细胞移植组,D组为左胫骨骨折+DMEM组,E组为T10脊髓横断+左胫骨骨折+ DMEM组,F组为左胫骨骨折+自体骨髓间充质干细胞移植组。模型建立2天后将BrdU标记的细胞移植到C、F组骨折断端。术后对B、C、E三组动物进行TARLOR评分,各组动物于术后1、2、3、4周拍摄左胫骨DR并检测、计算骨痂最大横截面。 结果:流式细胞仪检测发现BMSCs表达CD44阳性,CD34和CD45阴性,其染色前后表面标志物表达无明显统计学差异;BrdU染色标记的BMSCs最佳浓度和时间是10μmol/L、48 hours,DAPI染色标记BMSCs的最佳浓度和时间是20μg/ml、30 min,Hoechst33342染色标记BMSCs的最佳浓度和时间是5μg/ml、1 h。动物实验:术后B、C、E组TARLOR评分均达到实验要求;术后第1、2、3周,B、C、E组骨痂宽度较其余组有统计学意义(P<0.05),术后第4周,C组骨痂宽度较B、E组下降,但B、C、E组较其他组仍有统计学意义(P<0.05)。 结论:运用BrdU、DAPI 以及Hoechst33342三种方法标记BMSCs效果良好,方法可靠;大鼠骨髓间充质干细胞移植(BMSCs)改变了成骨潜能,促进了病理性骨痂向正常骨痂的转变,对失神经骨折愈合有积极正向作用。
Other AbstractObjective: To investigate the optimal dosage and timing for 5’-bromo-2’-deoxyuridine (BrdU),4_,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and hoechst 33342 labeling of rat bone marrow mesenchymal stem cells (BMSCs) in vitro and compare these three methods;To investigate the influence of autologous BMSCs transplantation on fracture healing combined with central nerve injuries in rats. Methods: BMSCs were isolated from the marrow of the tibia and the femur of male Sprague Daw-ley rat, the specific surface antigens (CD34, CD44, CD45) were identified by flow cytometry and BMSCs were labelled with BrdU, DAPI and hoechst 33342 respectively. The advantages and disadvantages of this three methods were analyzed. Cell proliferation was evaluated by using MTT. Then 48 healthy adult SD rats (male) were randomly divided into 6 groups (8 rats in each group): group A, simple(left) tibial fracture; group B, tibial fracture combined with T10 spinal cord transection (SCT) ; group C, tibial fracture combined with T10 spinal cord transection (SCT) + autologous BMSCs transplantation ; group D, tibial fractures + DMEM;group E, tibial fracture combined with T10 spinal cord transection (SCT) + DMEM , group F, tibial fracture combined with BMSCs transplantation. BMSCs were labeled with BrdU and implanted into the fractures of C、F groups when the model were made well after 2 days. B, C, E groups of animals were evaluated by using TARLOR score postoperation; The fractured tibiae were radioactivity(X-ray )evaluated separately at 1w, 2w, 3w and 4w after injury to assess fracture healing. Result: Flow cytometry showed that BMSCs expressed CD44 but not CD34 or CD45 and there was no significant difference in the expression of stem cell specific surface antigen between before labeling and after labeling. The optimal concentration and timing of the BMSCs with BrdU labeling were respectively 10 μmol/L and 48 hours and that of DAPI labeling were 20 μg/ ml and 30 min and 5 μg/ ml, 1 h with hoechst 33342. All of B, C, E groups animals TARLOR score reaches the requirement of experiment. 1, 2, 3 weeks after operation,the tibia callus width of B, C, E, groups are statistically significant (P < 0.05) than the rest of the groups;4 weeks the tibia callus width of group C decreased compared B and E groups, but B, C, and E groups are still statistical significance (P < 0.05) than the rest of the group. Conclusions: The results suggest that the three different ways of labeling ...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/224030
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
雷栓虎. MSCs的标记跟踪及其移植后对大鼠失神经骨折病理性骨痂塑形影响的实验研究[D]. 兰州. 兰州大学,2013.
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